Dual-Track Multifunctional Bimetallic Metal-Organic Frameworks for Antibiotic Enrichment and Detection.

The improper use and overuse of antibiotics have led to significant burdens and detrimental effects on the environment, food supply, and human health. Herein, a magnetic solid-phase extraction program and an optical immunosensor based on bimetallic Ce/Zr-UiO 66 for the detection of antibiotics are developed. A magnetic Fe3O4@SiO2@Ce/Zr-UiO 66 metal-organic framework (MOF) is prepared to extract and enrich chloramphenicol from fish, wastewater, and urine samples, and a horseradish peroxidase (HRP)-Ce/Zr-UiO 66@bovine serum protein-chloramphenicol probe is used for the sensitive detection of chloramphenicol based on the dual-effect catalysis of Ce and HRP. In this manner, the application of Ce/Zr-UiO 66 in integrating sample pretreatment and antibiotic detection is systematically investigated and the associated mechanisms are explored. It is concluded that Ce/Zr-UiO 66 is a versatile dual-track material exhibiting high enrichment efficiency (6.37 mg g-1) and high sensitivity (limit of detection of 51.3 pg mL-1) for chloramphenicol detection and serving as a multifunctional MOF for safeguarding public health and hygiene.

Evaluation of the antibacterial activity and protein profiling of Nile tilapia (Oreochromis niloticus) epidermal mucus under different feeds and culture systems (biofloc technology and earthen pond).

The mucus layers of fish serve as the main interface between the organism and the environment. They play an important biological and ecological role. The current study focuses on Nile tilapia epidermal mucus reared under different commercial feeds (coded A and B) and environments (biofloc technology and earthen pond systems). Crude protein levels in feed A and B were 30% and 28%, respectively. Water parameters in all culturing systems were suitable for tilapia throughout the study period. The antimicrobial potency of tilapia (n = 5 from each) epidermal mucus was tested in vitro against human and fish pathogenic strains viz. Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Francisella noatunensis, and Aeromonas hydrophila. To determine the antimicrobial activity, zones of inhibition (ZOI) were measured in millimetres and compared with two antibiotics (chloramphenicol and ciprofloxacin). SDS-PAGE analysis was performed on skin mucus samples of tilapia to determine protein quantity and size (molecular weight). Results of tilapia skin mucus (crude and aqueous) revealed a strong antibacterial effect against all the selected pathogenic strains. However, variation has been observed in the mucus potency and ZOI values between the biofloc and pond tilapia mucus. The crude mucus of tilapia fed on feed A and cultured in the pond exhibited strong antibacterial effects and high ZOI values compared to the mucus of biofloc tilapia, aqueous mucus extracts and positive control chloramphenicol (antibiotic). The SDS-PAGE results showed that the high molecular weight proteins were found in the collected epidermal mucus of BFT-B (240 kDa) and EP-B (230 kDa). Several peptides in fish skin mucus may play a crucial role in the protection of fish against disease-causing pathogens. Thus, it can be utilized in the human and veterinary sectors as an 'antimicrobial' for treating various bacterial infections.

Analysis and risk assessment of pharmaceutical residues in fish from three water bodies in Ghana.

Illegal mining has overshadowed pharmaceutical pollution even though exposure to pharmaceutical waste is high. Consumption of fish potentially polluted with pharmaceuticals from the rivers continues with little concern or potential threat it poses. In the present study, the residues of one antibiotic (Chloramphenicol), five hormones (progesterone, 17-beta Estradiol, Estrone, 17a-Ethynylestradiol, and one), three environmental contaminants (4-para-nonylphenol, 4-tert-octylphenol, and Bisphenol A), one barbiturate (Primidone) and one analgesic (Diclofenac sodium salt), were investigated from fish samples from the rivers Pra, Narkwa, and the Volta. The results show a high concentration of drugs in River Pra in comparison to those in Rivers Narkwa and Volta. The hazard quotients (HQs) for the environmental contaminants were all above 1, except Bisphenol A. Furthermore, the HQs from this study suggest that consumers of fish from any of the three rivers stand a hazard risk of Chloramphenicol (19), 17a-Ethynylestradiol (4), Estrone (1.366), Diclofenac sodium salt (3.29), Progesterone (4.598), 4-tert-octylphenol (87.2), and 4-para-nonylphenol (7.252), but negligible risk against E2 (0.687), Primidone (0.014), Testosterone (0.16), and Bisphenol A (0.642). Of the fish species studied, the highest concentration of all pharmaceuticals put together is found in Clarias gariepinus, Labeo senegalensis, and Chrysichthys nigrodigitatus in that order.

A graphene-oxide-based aptasensor for fluorometric determination of chloramphenicol in milk and honey samples utilizing exonuclease III-assisted target recycling and Nb.BbvCI-powered DNA walker cascade amplification.

Herein, a graphene oxide (GO)-based fluorescence aptasensor was developed for the sensitive and selective detection of chloramphenicol (CAP), based on exonuclease III (Exo III)-assisted target recycling and Nb.BbvCI-driven DNA walker cascade amplification. Interactions between CAP, hairpin1(HP1), hairpin2 (HP2), and 3'-amino modified hairpin3 (HP3) labeled with carboxyfluorescein (FAM) and covalently coupled to GO enabled efficient CAP detection. CAP was quantitatively assayed by measuring fluorescence at excitation/emission wavelengths of 480/514 nm, resulting from the accumulation of released FAM. A good linear range of 1 fM to 1 nM and a limit of detection (LOD) of 0.875 fM (signal-to-noise (S/N)= 3) were achieved. This aptasensor can distinguish the CAP from interference antibiotics with good specificity and selectivity, even if the concentration of the interfering substance is ten-fold higher than the target concentration. Moreover, the developed fluorescence aptasensor was successfully applied for the detection of CAP in spiked milk and honey samples. Thus, this method is potentially applicable for assaying CAP in foods and provides a promising strategy for the development of fluorescence aptasensors for environmental sample analysis.

Removal of chloramphenicol and resistance gene changes in electric-integrated vertical flow constructed wetlands.

The performance of an electric-integrated vertical flow constructed wetland (E-VFCW) for chloramphenicol (CAP) removal, changes in microbial community structure, and the fate of antibiotic resistance genes (ARGs) were evaluated. CAP removal in the E-VFCW system was 92.73% ± 0.78% (planted) and 90.80% ± 0.61% (unplanted), both were higher than the control system which was 68.17% ± 1.27%. The contribution of anaerobic cathodic chambers in CAP removal was higher than the aerobic anodic chambers. Plant physiochemical indicators in the reactor revealed electrical stimulation increased oxidase activity. Electrical stimulation enhanced the enrichment of ARGs in the electrode layer of the E-VFCW system (except floR). Plant ARGs and intI1 levels were higher in the E-VFCW than in the control system, suggesting electrical stimulation induces plants to absorb ARGs, reducing ARGs in the wetland. The distribution of intI1 and sul1 genes in plants suggests that horizontal transfer may be the main mechanism dispersing ARGs in plants. High throughput sequencing analysis revealed electrical stimulation selectively enriched CAP degrading functional bacteria (Geobacter and Trichlorobacter). Quantitative correlation analysis between bacterial communities and ARGs confirmed the abundance of ARGs relates to the distribution of potential hosts and mobile genetic elements (intI1). E-VFCW is effective in treating antibiotic wastewater, however ARGs potentially accumulate.

Sources, Environmental Fate, and Ecological Risks of Antibiotics in Sediments of Asia's Longest River: A Whole-Basin Investigation.

This study conducted the first extensive and comprehensive investigation of the whole-scale sedimentary antibiotic concentration, possible drivers, environmental fate, and potential ecological risks in the Yangtze River. Totally, 20 antibiotics were detected in the sediments. Results revealed that the order of antibiotic abundance in sediment was fluoroquinolones > tetracyclines > macrolides > sulfonamides > amphenicols. The total antibiotic concentrations were 0.10-134.4 ng/g (mean: 11.88 ng/g). Of these, fluoroquinolones and tetracyclines were the two dominant antibiotic categories. The dominant occurrence of fluoroquinolones and tetracyclines in sediments suggested that the distribution coefficient (Kd) was one of the important factors to determine their fate. Correlation analysis demonstrated that antibiotic contamination was largely influenced by the local scale of animal husbandry, and the positive correlation between antibiotics and heavy metals was likely driven by their common source of contamination and the complexation. Environmental risk assessment showed that tetracycline and chlortetracycline exhibited potential risks from medium to high in the Yangtze River, although most of the compounds posed minimal and low risks. This work provided a valuable large-scale data set across the whole Yangtze River and revealed the contamination profile of antibiotics. Mitigation and management measures to reduce antibiotic inputs are needed for the Yangtze River basin.

Novel and simple analytical method for simultaneous determination of sulfonamide, quinolone, tetracycline, macrolide, and chloramphenicol antibiotics in soil.

The multiclass determination of antibiotic residues in the soil is challenging because of its complex physicochemical properties. In this study, a simple analytical method was developed to simultaneously extract and determine 58 antibiotics from the soil. A novel acidity-regulated extraction-partition-concentration protocol was established for the simultaneous extraction of five classes (23 sulfonamides, 18 quinolones, five tetracyclines, eight macrolides, and four chloramphenicols) of antibiotics from the soil. Compared to traditional methods, the sample preparation efficiency was significantly improved by four times (45 min vs. 230 min) by optimizing the extraction method and omitting the time-consuming solid-phase extraction (SPE) procedure. The ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was optimized to determine the 58 antibiotics in a single run by applying positive/negative switching acquisition mode in less than 10 min with the baseline separation of sulfameter and sulfamethoxypyridazine. Suitable recoveries, ranging between 60 and 120%, were obtained for most antibiotics, with RSD <20%. The limits of quantification (LOQ) of the method were 2 µg/kg and 5 µg/kg. Thus, this study provides a simple, reliable, and economical method for accurately and rapidly determining a multiclass of antibiotics in the soil.

MXene-AuNP-Based Electrochemical Aptasensor for Ultra-Sensitive Detection of Chloramphenicol in Honey.

A simple and label-free electrochemical aptasensor was developed for ultra-sensitive determination of chloramphenicol (CAP) based on a 2D transition of metal carbides (MXene) loaded with gold nanoparticles (AuNPs). The embedded AuNPs not only inhibit the aggregation of MXene sheets, but also improve the quantity of active sites and electronic conductivity. The aptamers (Apts) were able to immobilize on the MXene-AuNP modified electrode surface through Au-S interaction. Upon specifically binding with CAP with high affinity, the CAP-Apt complexes produced low conductivity on the aptasensor surface, leading to a decreased electrochemical signal. The resulting current change was quantitatively correlated with CAP concentration. Under optimized experimental conditions, the constructed aptasensor exhibited a good linear relationship within a wide range of 0.0001-10 nM and with a low detection limit of 0.03 pM for CAP. Moreover, the developed aptasensor has been applied to the determination of CAP concentration in honey samples with satisfactory results.

Urinary concentrations of amphenicol antibiotics in relation to biomarkers of oxidative DNA and RNA damage in school children.

Previous studies implied that elevated exposure to amphenicol antibiotics may induce increased oxidative stress. However, the effects of amphenicol antibiotics exposure on oxidative stress damage in human have not been well studied. This study examined the associations between amphenicol antibiotics exposure and oxidative damage biomarkers in school children. Three major amphenicols including chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF) and two biomarkers of 8-hydroxydeoxyguanosine (8-OHdG) for oxidative DNA damage and 8-oxo-7,8- dihydroguanosine (8-OHG) for oxidative RNA damage were measured in 414 morning urine samples collected from 70 school children in Shanghai, China. School children were exposed to CAP, TAP, and FF with median concentrations of 1.37, 0.36, and 0.06 µg/g Cre, respectively. Linear mixed models revealed that an interquartile range (IQR) increase of urinary TAP was positively associated with 7.75%(95% CI: 4.40%, 11.1%) increase of 8-OHdG and 7.48%(95% CI: 2.49%, 15.6%) increase of 8-OHG, respectively; in addition, CAP was associated with elevated 8-OHdG. Although FF was not found to be significantly associated with either 8-OHdG or 8-OHG, it is warranted to further investigate FF and its metabolites levels in relation to oxidative stress in future study. Our findings provide new evidence for the effects of exposure to TAP and CAP on nucleic acid oxidative damage in Children.

Radiotracer studies to isolate in-house receptors from poultry liver for multi-chemical hazard analysis in selected food and feed.

In-house receptors (IHRs) were isolated from non-immunized poultry liver to analyze selected contaminants and residues in targeted food and feed using 14C- and 3H-labeled radiotracers. Matrix (2 g) was homogenized and centrifuged with the resultant pellet used as IHRs. These were characterized for total protein contents (6.1 mg mL-1) and compared with commercial receptors for aflatoxins (0.28 mg tablet-1) and chloramphenicol (0.12 mg tablet-1). Gel electrophoresis of the IHRs showed a mixture of polypeptides-an important attribute for multi-residues analysis-compared with commercial receptors that presented specific protein bands at 65 kDa (chloramphenicol) and 70 kDa (aflatoxins). The inhibition index of IHRs for aflatoxins B1 and B2 in wheat and bovine feed and chloramphenicol in bovine tissue at, above, and below maximum limits or minimum required performance limits, revealed an inverse relationship between radiotracer and analyte concentrations. Saturation with increased radioligand concentration up to 5.5 kBq indicated higher holding potential. However, increasing incubation time to 30 min did not significantly increase analyte-binding. The IHRs performance was comparable to commercial receptors with control point averages of 348, 410, 555, and 307 counts per minute determined for gentamicin, chloramphenicol, oxytetracycline, and aflatoxin M1, respectively in local milk samples.

Aptamer Self-Assembly-Functionalized Nanochannels for Sensitive and Precise Detection of Chloramphenicol.

Sensitive and precise determination of chloramphenicol (CAP) is of great significance for human health due to its high risk in trace amounts. Solid-state artificial nanochannels are expected to be highly promising sensing devices owing to single-molecule sensitivity, target-specific selectivity, and portability. Herein, we report an aptamer self-assembly-functionalized artificial nanochannel-based sensor for highly sensitive and precise determination of CAP. Aptamer self-assembly (AAs) served as the specific recognition component and were in situ grown on the surface of stable anodic aluminum oxide (AAO) nanochannels to develop an AAs@AAO nanochannel-based sensor. Selective interaction with CAP led to the disassembly of AAs and sensitive current change of AAs@AAO nanochannels, allowing sensitive and precise sensing of CAP in complex food samples. The developed AAs@AAO nanochannel-based sensor showed a wide linear range from 0.32 to 1600 pg. mL-1, low limit of detection (LOD) of 0.1 pg. mL-1, high precision with relative standard deviation of 2.9%, and quantitative recoveries of 93.4-102.2% for CAP in milk, milk powder, and honey samples. This work proposes a versatile nanochannel-based platform for facile, sensitive, and precise sensing of hazardous residues in food samples.

Synthesis of molecularly imprinted microspheres and development of a fluorescence method for detection of chloramphenicol in meat.

In this study, nitrobenzene was used as dummy template to synthesize a type of specific molecularly imprinted microspheres for chloramphenicol, and 4-nitroaniline was coupled with three fluorophores to synthesize three fluorescent tracers. Then a competitive fluorescence method was developed on a conventional microplate for detection of chloramphenicol in chicken and pork samples. This method contained only one sample-loading step, so one assay was finished within 30 min. The IC50 was 1.8 ng/ml, and the limit of detection was 0.06 ng/g. The recoveries from chloramphenicol-fortified blank meat samples were in the range 67.5-96.2%. Furthermore, this method could be recycled three times. The detection results for some real meat samples were identical to that of a LC-MS/MS method. Therefore, this method could be used as a practical tool for routine screening for the residue of chloramphenicol in large number of meat samples.

An electrochemical aptasensor based on PEI-C3N4/AuNWs for determination of chloramphenicol via exonuclease-assisted signal amplification.

An electrochemical aptasensor, including the polyethyleneimine-graphite-like carbon nitride/Au nanowire nanocomposite (PEI-C3N4/AuNWs) and exonuclease-assisted signal amplification strategy was constructed for the determination of chloramphenicol (CAP). Initially, a nanocomposite with substantial electrocatalytic property was synthesized by PEI-C3N4/AuNWs. This improves the conductivity and specific surface area of the PEI-C3N4/AuNW-modified gold electrode. Next, a DNA with a complementary sequence to a CAP aptamer (cDNA) was immobilized on the PEI-C3N4/AuNW-modified electrode, followed by the CAP aptamer hybridized with cDNA. The lower signal at this time is due to the negatively charged phosphate group of the oligonucleotide and [Fe (CN)6]3-/4- electrostatically repelling each other. The presence of the CAP would cause aptamer on the electrode surface to fall off and be digested by Recjf exonuclease, which resulted in target recycling, and a significant increase in DPV signal can be observed at a potential of 0.176 V (vs. Ag/AgCl). Under optimal conditions, there is a linear relationship between the peak current and the logarithm of CAP concentration in the range 100 fM-1 µM, and the detection limit of this aptasensor is 2.96 fM (S/N = 3). Furthermore, the resultant aptasensor has excellent specificity, reproducibility, and long-term stability, and has been applied to the detection of CAP in milk samples. Graphical abstract The detection principle of the electrochemical aptasensor for CAP detection was based on PEI-C3N4/AuNWs and exonuclease-assistant signal amplification. It is based on the fact that PEI-C3N4/AuNWs nanocomposites on the surface of the electrode can effectively improve the performance of the aptasensor, and Recjf exonuclease initiates the target recycling process, causes signal amplification.

Intertwined Carbon Nanotubes and Ag Nanowires Constructed by Simple Solution Blending as Sensitive and Stable Chloramphenicol Sensors.

Chloramphenicol (CAP) is a harmful compound associated with human hematopathy and neuritis, which was widely used as a broad-spectrum antibacterial agent in agriculture and aquaculture. Therefore, it is significant to detect CAP in aquatic environments. In this work, carbon nanotubes/silver nanowires (CNTs/AgNWs) composite electrodes were fabricated as the CAP sensor. Distinguished from in situ growing or chemical bonding noble metal nanomaterials on carbon, this CNTs/AgNWs composite was formed by simple solution blending. It was demonstrated that CNTs and AgNWs both contributed to the redox reaction of CAP in dynamics, and AgNWs was beneficial in thermodynamics as well. The proposed electrochemical sensor displayed a low detection limit of up to 0.08 µM and broad linear range of 0.1-100 µM for CAP. In addition, the CNTs/AgNWs electrodes exhibited good performance characteristics of repeatability and reproducibility, and proved suitable for CAP analysis in real water samples.

Single and ternary competitive adsorption-desorption and degradation of amphenicol antibiotics in three agricultural soils.

The widespread usage of veterinary antibiotics results in antibiotic contamination and increases environmental risks. This study was evaluated the single and ternary competitive adsorption-desorption and degradation of three amphenicol antibiotics (AMs): chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FF) in three agricultural soils. The adsorption capacity of amphenicol antibiotics in the soil was weak, and the Kf value was in the range of 0.15-3.59 µg1-1/nL1/n kg-1. In the single adsorption-desorption experiment, the ranked order of adsorption capacity was TAP > FF > CAP. However, in the ternary competitive adsorption experiment, the order was changed to be CAP > FF > TAP. The degradation of AMs in soils was performed at various conditions. All AMs were vulnerable to microbial degradation in soils. A higher initial concentration would reduce the degradation rate and enhance the persistence of AMs in soil. The degradation of AMs was positively influenced by changes in soil moisture content and culture temperatures up to 30 °C and decreased at higher temperatures. An equation was used to predict the leachability of AMs in soils and assess their risk to the water environment. The weak adsorption capacity and poor persistence of FF indicated that it may have a strong effect on groundwater based on the equation. It is imperative to further assess the biological impacts of FF at environmentally relevant concentrations given its mobility and extensive use in the livestock industry.

Molecularly Imprinted Silica-Coated CdTe Quantum Dots for Fluorometric Determination of Trace Chloramphenicol.

A dual recognition system with a fluorescence quenching of quantum dots (QDs) and specific recognition of molecularly imprinted polymer (MIP) for the detection of chloramphenicol (CAP) was constructed. MIP@SiO2@QDs was prepared by reverse microemulsion method with 3-aminopropyltriethoxysilane (APTS), tetraethyl orthosilicate (TEOS) and QDs being used as the functional monomer, cross-linker and signal sources, respectively. MIP can specifically recognize CAP, and the fluorescence of QDs can be quenched by CAP due to the photo-induced electron transfer reaction between CAP and QDs. Thus, a method for the trace detection of CAP based on MIP@SiO2@QDs fluorescence quenching was established. The fluorescence quenching efficiency of MIP@SiO2@QDs displayed a desirable linear response to the concentration of CAP in the range of 1.00~4.00 × 102 µmol × L-1, and the limit of detection was 0.35 µmol × L-1 (3σ, n = 9). Importantly, MIP@SiO2@QDs presented good detection selectivity owing to specific recognition for CAP, and was successfully applied to quantify CAP in lake water with the recovery ranging 102.0~104.0%, suggesting this method has the promising potential for the on-site detection of CAP in environmental waters.

Dynamic light scattering biosensing based on analyte-induced inhibition of nanoparticle aggregation.

A new approach to direct quantitative detection of small molecules (haptens) by dynamic light scattering biosensing is presented. The proposed technique implements a homogeneous competitive immunoassay and is based on optical detection of specific inhibition of nanoparticle aggregation induced by the analyte in a sample. The technique performance was tested both in buffer and milk for detection of chloramphenicol - antibiotic relevant to food safety diagnostics. Good specificity, sensitivity (LOD in milk is 2.4 ng/ml), precision (4.0 ± 1.2%), ruggedness (8.3%), and 96% recovery in conjunction with a record wide dynamic range (3 orders of magnitude) of the nanosensing technique were demonstrated. Such characteristics complemented by the assay simplicity (no washing step) and a short assay time make the approach attractive for application as an analytical platform for point-of-care and field-oriented diagnostics. Graphical abstract.

Covalently bonded aptamer-functionalised magnetic mesoporous carbon for high-efficiency chloramphenicol detection.

A novel aptamer-modified magnetic mesoporous carbon was prepared to develop a specific and sensitive magnetic solid-phase extraction method through combination with ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis chloramphenicol in complex samples. More specifically, the chloramphenicol aptamer-modified Mg/Al layered double hydroxide magnetic mesoporous carbon was employed as a novel magnetic solid-phase extraction sorbent for analyte enrichment and sample clean-up. The extraction solvent, extraction time, desorption solvent, and desorption time were investigated. It was found that the mesoporous structure and aptamer-based affinity interactions resulted in acceptable selective recognition and a good chemical stability toward trace amounts of chloramphenicol. Upon combination with the ultra-high performance liquid chromatography-tandem mass spectrometry technique, a specific and sensitive recognition method was developed with a low limit of detection (0.94 pmol/L, S/N = 3) for chloramphenicol analysis. The developed method was successfully employed for the determination of chloramphenicol in complex serum, milk powders, fish and chicken samples, giving recoveries of 87.0-107% with relative standard deviations of 3.1-9.7%.

Electrochemical determination of chloramphenicol and metronidazole by using a glassy carbon electrode modified with iron, nitrogen co-doped nanoporous carbon derived from a metal-organic framework (type Fe/ZIF-8).

In this study, an iron-doped metal-organic framework (MOF) Fe/ZIF-8 was synthesized from ZIF-8 at room temperature. Direct carbonization of Fe/ZIF-8 under a nitrogen atmosphere produced nanoporous nitrogen doped carbon nanoparticles decorated with Fe component (Fe/NC). The Fe/NC exhibited a large surface area (1221.185 m2 g-1) and narrow pore-size distribution (3-5 nm). The nanoporous Fe/NC components along with Nafion were used to modify a glassy carbon electrode for the electrochemical determination of chloramphenicol and metronidazole via linear sweep voltammetry. Under optimal conditions, the reduction peak currents (observed at -0.237 V and -0.071 V vs. Ag/AgCl) of these analytes increased linearly with increasing chloramphenicol and metronidazole concentrations in the range of 0.1-100 µM and 0.5-30 µM, with the detection limits estimated to be 31 nM and 165 nM, respectively. This result was attributed to the large surface area, porous structure, high nitrogen content, and as well as the electrocatalytic effect of Fe atoms embeded in the carbon support. The proposed sensor was used for chloramphenicol and metronidazole analysis in samples, providing satisfactory results.

Detection of chloramphenicol with an aptamer-based colorimetric assay: critical evaluation of specific and unspecific binding of analyte molecules.

A chloramphenicol (CAP)-binding aptamer of 80 nucleotides (nt) was reported in 2011. In 2014, it was truncated to 40 nt and has since been used by most researchers, although a careful binding study is still lacking. In this work, binding assays using isothermal titration calorimetry and various DNA-staining dyes were performed. By comparing the truncated aptamer with three control sequences, no specific binding of CAP was observed in each case. The secondary structures of the original and truncated aptamers were analyzed, and it was shown that the likelihood of the truncated aptamer to retain the same binding mechanism as the original sequence is low. We further examined gold nanoparticle (AuNP)-based label-free colorimetric assays. By quantifying the extinction ratio at 620 nm over that at 520 nm, a similar color response was observed regardless of the sequence of DNA, suggesting the color change mainly reflected other events such as the adsorption of CAP by the AuNPs, instead of aptamer binding to CAP. Salt-induced aggregation experiments suggested direct adsorption of CAP on AuNPs. CAP only weakly inhibited DNA adsorption by AuNPs but did not displace pre-adsorbed DNA. Therefore, CAP adsorption by AuNPs needs to be considered when designing related sensors, for example, by using non-aptamer sequences as controls. This work calls for careful confirmation of aptamer binding and control experiments for designing aptamer and AuNP-based biosensors.